Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside HIV-1 RT inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.05.040

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent an integral part of the currently available combination antiretroviral therapy (cART) contributing to reduce the AIDS-mortality and turned the disease from lethal to chronic. In this conte

Full text of DOI:10.1016/j.bmc.2017.05.040

Accepted Manuscript
Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside
HIV-1 RT inhibitors
Stefania Ferro, Maria Rosa Buemi, Laura De Luca, Fatima E. Agharbaoui,
Christophe Pannecouque, Anna-Maria Monforte
PII:
S0968-0896(17)30476-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.040
BMC 13758
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 March 2017
9 May 2017
17 May 2017
Please cite this article as: Ferro, S., Buemi, M.R., Luca, L.D., Agharbaoui, F.E., Pannecouque, C., Monforte, A-M.,
Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside HIV-1 RT inhibitors, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.040
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Graphical Abstract
Leave this area blank for abstract info.
Searching for novel N1-substituted
benzimidazol-2-ones as non-
nucleoside HIV-1 RT inhibitors
a
a
a
a
Stefania Ferro, Maria Rosa Buemi, Laura De Luca, Fatima E. Agharbaoui, Christophe
Pannecouque, b and Anna Maria Monfortea*
aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina,
b
98168 Messina, Italy; Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000
Leuven, Belgium
R
n
O
S
N
O
Cl
O
N
H

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside HIV-1
RT inhibitors
Stefania Ferro, a Maria Rosa Buemi, a Laura De Luca, a Fatima E. Agharbaoui, a Christophe Pannecouque, b and
Anna-Maria Monforte a*
aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Università degli Studi di Messina, Viale Annunziata, 98168
Messina, Italy
b
KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent an integral part of the
currently available combination antiretroviral therapy (cART) contributing to reduce the AIDS-
mortality and turned the disease from lethal to chronic. In this context we recently reported a
series of 6-chloro-1-(3-methylphenylsulfonyl)-1,3-dihydro-2Hbenzimidazol-2-ones as potent
non-nucleoside HIV-1 reverse transcriptase inhibitors. In this paper, we describe the design and
the synthesis of two novel series of benzimidazolone analogues in which the linker moiety
between the phenyl ring and the sulfonyl group was modified and new small lipophilic groups
on the benzyl sulfonyl pendant were introduced. All the new obtained compounds were
evaluated as RT inhibitors and were also tested against RTs containing single amino acid
mutations. Finally, molecular docking studies were performed in order to rationalize the
observed activity of the most promising compound.
Available online
Keywords:
Reverse Transcriptase Inhibitors
Design
Synthesis
Benzimidazolones
RTs mutant
2009 Elsevier Ltd. All rights reserved
.
Introduction
Reverse transcriptase (RT) enzyme is a crucial target for inhibition of HIV-1 infection and non-nucleoside reverse transcriptase
inhibitors (NNRTIs) represent an integral part of the currently available combination antiretroviral therapy (cART).1-3 Although the
cART dramatically reduced the AIDS-mortality and turned the disease from lethal to chronic, it is not able to completely eradicate the
virus from the body requiring a lifetime use.4 Moreover, drug resistance and severe side effects such as dyslipidemias, insulin
resistance, lipodystrophy, heart diseases and other related disorders 5-7 are still the main reasons of the therapy failure.8-10 NNRTIs bind
a specific hydrophobic region of the enzyme which is a flexible pocket formed upon the binding with the inhibitor. By mutational
studies some amino acids (Trp229, Phe227, Leu234 and Tyr318) of non-nucleoside inhibitor binding pocket (NNIBP) have been found
to play an important role in proper binding resulting less prone to mutation.11, 12 So, a possible strategy for the design of novel inhibitors
can be addressed to target this highly conserved area where the lowest tendency to acquire mutations is expected.
In addition, the flexibility of the binding pocket in RT accounts for the structural and chemical diversity of NNRTIs, providing wide
opportunities for novel scaffolds discovery.13, 14 Moreover, according to this idea a powerful inhibition of the mutant variants of HIV-1
could be linked to the ability of the molecule to adopt variable binding conformations. 15
In previous papers we reported several N1-substituted 6-chloro-1,3-dihydro-2H-benzimidazol-2-ones 16-18 as potent HIV-1 NNRTIs,
and among them some derivatives proved to be less toxic and more active than nevirapine and, in some cases, than efavirenz, two
currently clinically used compounds (Figure 1).

O
H
N
H
N
O
O
Cl
N
N
N
CF3
b
a
RR'
X
N
RR'
Y
X=CH2, SO2
Y= O, S
N
H
c
Figure 1: Chemical structure of a) efavirenz, b) nevirapine and c) N1-substituted 6-chloro-1,3-dihydro-2H-benzimidazol-2-ones
Extended structure–activity relationships (SAR) studies highlighted that the nature and the position of the substituents on the phenyl
ring at N1 of the benzimidazolone system significantly influenced the anti-HIV-1 activity. Moreover, molecular modeling studies
performed on this class of compounds by us emphasized the beneficial contribution to the antiviral activity due to the introduction of a
chlorine atom on the benzimidazole system as well as the presence of the sulfonyl bridge, able to form intermolecular interactions with
important residues of the NNIBP.18
Considering these relevant findings and the structural features proving to be fundamental in the enzymatic inhibitors active against
NNRTIs-resistant mutants, we herein report the design and the synthesis of new N1-aryl-benzimidazol-2-ones with the aim of
improving the antiretroviral activity of this class of compounds and obtaining new useful insights for their structure activity
relationships. Finally, molecular docking studies are performed to clarify the binding mode of the most promising derivatives.
Results and discussion
Rational design
Conformational flexibility and positional adaptability of NNRTIs can contribute to increase the inhibitors efficacy against a variety
of drug-resistant HIV-1 strains. 19 In the classical concept of drug design, a favorable inhibitor should efficiently enter and maximally
occupy the binding site, thus interacting effectively with the residues around the binding pocket and improving the potency of NNRTIs.
In this context, a new benzimidazolone derivative with a bulky substitution at N1, 2-{6-bromo-2-fluoro-3-[(2-oxo-2,3-dihydro-1H-
20
benzimidazol-1-yl)methyl]phenoxy}-4-chlorobenzonitrile (A), has been reported
as promising HIV-1 NNRT inhibitor, showing
activity at nanomolar level (Figure 2).
NC
O
Cl
F
Br
N
O
N
H
Figure 2: Chemical structure of 2-{6-bromo-2-fluoro-3-[(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)methyl]phenoxy}-4-chlorobenzonitrile (A)
16
Furthermore, in our previously reported paper
we described the 6-chloro-1-(3-methylphenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (B) (Figure 3) as a potent non-nucleoside HIV-1 reverse transcriptase inhibitor.
CH3
SO2
Cl
N
O
N
H
Figure 3: Chemical structure of 6-chloro-1-(3-methylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-2-one (B)
In order to better understand the behavior of benzimidazolone derivatives A and B, they were both flexibly docked into the NNIBP
21
22
of HIV-1 RT. Autodock Vina and the reported X-ray structure of the complex RT/GW564511 (PDB ID: 3DLG)
were used,
following a new docking procedure performed by us (see experimental part).

Figure 4 (I) shows the possible binding mode of the reported compound A. Particularly, the benzimidazolone scaffold seems able to
form two hydrogen bonds between NH and oxygen of the imidazolone nucleus and oxygen and NH of the Lys103, respectively. In
addition, the phenyl group of compound A, fluoro-bromo substituted, is planted in the center of the binding pocket. In figure 4 (II) the
binding mode of compound B, appearing well housed into the hydrophobic pocket delimited by residues of Val106, Tyr181, Tyr188
and Trp229, is shown. Particularly, we highlighted the creation of a hydrophobic contact between the phenyl sulfonyl moiety of
compound B and the highly conserved residue of Trp229, required for an efficient viral replication. Moreover an interaction between
the sulphonyl bridge and the residue of Tyr181 was predicted. Finally, the nitrogen of the benzimidazolone nucleus establishes a
hydrogen bond with the key amino acid Lys101 conferring more stability to the complex.
Taking into account the results obtained by means of our new docking studies and as an extension of our ongoing efforts towards the
development and identification of new molecules with anti HIV-1 activity, we herein report the structural modification of our reference
compound B and the synthesis of novel N1-substituted benzimidazolones, characterized by the introduction of a more flexible and
malleable portion at N1.
Figure 4: Binding mode of compound A (I) and B (II) in complex with RT. Key residues of the NNIBP are presented. The hydrogen bonds are in dotted lines.
More specifically, we decided to synthetize new benzimidazolone analogues 6 (a-g) and 9 (a-g) in which, while in the view of their
paramount importance, the sulfonyl moiety at N1 and the chlorine atom at 6 position of the system were kept unchanged, a methylene or
ethylene bridge between the phenyl ring and the sulfonyl group has been inserted (Figure 5).
The hypothesis was that the greater flexibility of the introduced moiety might provide an advantage in the potency of the final
compounds.
R
n
O
S
N
O
Cl
O
N
H
n= 1 or 2
Figure 5: Chemical structure of the new designed N1-substituted benzimidazol-2-ones.
Furthermore in order to improve the hydrophobic contacts, we also analyzed the effects on the antiretroviral activity due to the
addition, on the different positions of the benzyl sulfonyl moiety, of small lipophilic groups, such as chlorine atom or methyl
functionality.
Chemistry
The designed compounds 6 (a-g) and 9 (a-g) have been synthesized according to the synthetic pathway reported in Scheme 1.
Commercially available 5-chloro-2-nitro-aniline was N-alkylated by treatment with appropriate substituted benzensufonyl chloride 1
(a-g) or 2-phenylethanesulfonyl chloride 3 (a-g) using DMF as solvent in alkaline medium. To obtain compounds 9 (a-g), structurally
characterized by ethylene bridge, 2-phenylethanesulfonyl chlorides 3 (a-g), not commercially available, are required as reagents. They
were synthesized by treatment of the corresponding phenylethanbromide 2 (a-g) with KSAc in MeCN at 0°C for 1h; then HCl conc and
NaClO2 were added. The obtained nitro derivatives 4 (a-g) and 7 (a-g) were easily reduced using Zn dust in acidic medium. Finally, the
cyclization of amino derivatives 5 (a-g) and 8 (a-g) with COCl2 afforded the desired final compounds (6 and 9 a-g) in good yields.
Both analytical and spectral data (1H NMR) of all synthesized compounds were in full agreement with the proposed structures.

Scheme 1
Cl
O
R
O
S
a
H
R
R
R
b
c
2-Cl
3-Cl
4-Cl
d
e
n
n
n
R
Cl
NH2
NO2
2-CH3
3-CH3
4-CH3
O
S O
O
S
N
O
O
S O
NH
1a-g
f
Cl
Cl
NH
Cl
g
iv
iii
O
Cl
O
ii
N
H
O
S
Br
NH2
NO2
6 a-g n=1
9 a-g n=2
4 a-g n=1
7 a-g n=2
5 a-g n=1
8 a-g n=2
i
R
2 a-g
R
3 a-g
Reagents and Conditions: i) MeCN, KSAc 0°C, r.t. 1h; HClconc, NaClO2 r.t., 40’; ii) NaH, DMF, 0°C, 2-6 h; iii) HCl, EtOH, Zn 0°C, 100°C 1h; iv) COCl2,
HCl, H2O, 100°C, 4h.
Biological Activity
All the new synthesized derivatives 6 (a-g) and 9 (a-g) were tested for their ability to inhibit the enzymatic polymerase activity of
HIV-1 RT and the obtained results, summarized in Table 1, are compared to the FDA-approved drugs Nevirapine and Efavirenz.
Preliminary SAR analysis allowed us to highlight the effects on enzymatic inhibition due to the linker placed between the phenyl
ring and the sulfonyl group. Particularly, as we can see by comparison of the biological results of compounds 6 (a-g) and 9 (a-g), the
introduction of an extra methylene seems to be detrimental for the activity (ethylene vs methylene linker). In fact, compounds
belonging to the most promising 6-chloro-1-[(substituted-benzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-ones series 6 (a-g) exhibited
moderate to good activities ranging from 0.79 µM to 21 µM. However, we are not able to highlight any correlation between the
observed anti-RT effects and the nature and position of the substituents on the benzylsulfonyl moiety at-N1.
In addition, among compounds 6 (a-g) derivatives 6b and 6f showed the best activity with IC50 values of 1.3 µM and 0.79 µM
respectively, superior to that of reference compound nevirapine (IC50=1.55 µM).
Table 1. Anti-RT activity of compounds 6 (a-g) and 9 (a-g) and reference compound B.
Compound
IC50 (µM)a
Compound
IC50 (µM) a
6a
6b
17 5
1.3 0.0
6 0.5
18 3.9
9a
9b
9c
9d
9e
9f
76 27
116 34
131 34
95 18
29 3
6c
6d
6e
21 0.9
6f
6g
Bb
0.79 0.07
2.25 0.01
0.021 0.002
0.019 0.003
39 8
218 85
9g
Efavirenz
Nevirapine
1.55 0.25
aConcentration required to inhibit by 50% the in vitro RNA-dependent DNA polymerase activity of recombinant RT. bref [16].
Newly prepared derivatives 6 (a-g) and 9 (a-g) were also tested in MT-4 cells for inhibition of HIV-1 (strain IIIB); moreover compound-
induced cytotoxicity was measured in MT-4 cells in parallel with antiviral activity.
As listed in Table 2, analysis of obtained experimental data confirmed the same trend observed in the case of RT inhibition
considered that, once again, compounds structurally characterized by methylene bridge 6 (a-g) were significantly more active than the
corresponding derivatives in which ethylene linker was inserted 9 (a-g).
It can be observed that all the newly synthesized 6-chloro-1-[(substituted-benzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-ones
derivatives 6 (a-g) had an inhibitory effect on the cell at micromolar or nanomolar concentrations.
The more potent derivatives 6a, 6b, 6c, 6f and 6g inhibit HIV-1 replication at EC50 values ranging from 0.08 µM to 1.93 µM and
selectivity indices (SI) ranging from 48 to 1376.
Particularly, among them 6-chloro-1-[(3-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6f), 3-methyl substituted on
the benzyl sulfonyl moiety had antiviral activity in the cell assay at 0.08µM and a selectivity index greater than 1000 (SI=1376).
Interestingly,
this
compound
proved
to
be
also
the
best
enzymatic
inhibitor
(IC50=
0.79µM).

Table 2. Anti-HIV-1 activity, cytotoxicity and selectivity index in MT-4 cells of compounds 6 (a-g) and 9 (a-g) and reference
compound B.
Compound
EC50 (µM)a
CC50 (µM)b
SIc
Compound
EC50 (µM) a
CC50 (µM)b
SIc
6a
6b
0.83 0.28
0.19 0.01
1.14 0.05
5.06 0.41
10 1
0.08 0.01
1.93 0.09
0.0151 0.004
0.004 0.001
155 19
52 9
187
271
134
15
24
1376
48
>20528
>1316
9a
9b
9c
9d
9e
9f
> 30
> 100
> 64
> 91.
> 32
> 31
30 3
<1
<1
<1
<1
<1
<1
<1
100 37
63 28
91 38
32 3
31 3
161 9
6c
6d
152 12
77 29
238 20
110 13
93 25
>308
6e
6f
6g
Bd
9g
> 160
Efavirenz
>6.33
Nevirapine
0.18 0.006
>15.02
>81
aEffective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells. bCytotoxic concentration required to reduce MT-4 cell
viability by 50%. cSelectivity index: ratio CC50/EC50 d ref [16].
.
Docking studies
In order to explore the plausible binding mode of methylene 6(a-g) and ethylene 9(a-g) bridge series of compounds, comparative
docking studies of derivatives 6f and 9f into the non-nucleoside binding site of HIV-1 RT were performed, using AutoDock Vina
software. 21
The selected ligands were built by means of Discovery Studio 2.5.5 23 version and minimized using CHARMm forcefield followed
by Smart Minimizer algorithm in order to eliminate bad contacts and generate more stable poses. Docking of compounds 6f and 9f into
RT allosteric pocket generated an ensemble of possible binding conformations. After analyzing the output clusters, the most
energetically favorable conformation was chosen for further analysis.
Concerning compound 6f, the analysis of the obtained output clusters revealed a predominant orientation of the ligand. In Figure 6
the best conformation is displayed. As we can see by comparing Figure 4 (II) and Figure 6, derivative 6f interacts with the hotspot
amino acids of the allosteric pocket in a similar fashion to the reference compound B.
Figure 6: Binding mode of 6-chloro-1-[(3-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6f) in complex with RT. Key residues of the pocket
are presented. Hydrogen bonds are showed by dotted lines.
Particularly, the 3-methylphenyl group of 6f as well as the 3-methylphenyl moiety of compound B are well accommodated into the
hydrophobic pocket made by the residues of Tyr181, Tyr188 and Trp229. Furthermore for both derivatives (6f and B) the NH group of
the imidazolone frame establishes a hydrogen bond with the oxygen of residue of Lys101.
In Figure 7 the binding mode and the hydrophobic contacts of compound 9f are reported.

Figure 7: Binding mode of compound 6-chloro-1-[2-(3-methylphenyl)ethanesulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (9f) in complex with RT. Key
residues of the pocket are presented. Hydrogen bonds are shown by dotted lines.
By analysis of the output clusters no main orientation of the ligand was observed, albeit the most energetically favorable
conformation was chosen for further analysis.
We can suppose that the enzymatic inhibition of this compound (IC50=39 µM) is due to its ability to form a hydrogen bond with the
oxygen of the Lys101 through the NH group of the imidazolone, while the 3-methylphenyl group is engaged in hydrophobic contacts
with different amino acids located into the allosteric pocket. Finally the loss of the π-π interaction with Tyr188 could clarify its reduced
activity.
HIV-1 RT mutant studies
All the new compounds were tested against RTs containing different single amino acid mutations responsible for resistance to NNRTIs.
As shown in Table 3 the analysis of performed biological screening showed an unclear trend of the structure activity relationships,
however in many cases (L100I: 6b, 6f; K103N: 6f, 9e; Y181C: 6f, 6c, 9b-g; Y188L: 6g, 9d-g; G190A: 6a, 6c, 6f, 9b-g) an improved
inhibition profile in comparison with the reference compound Nevirapine (b) was observed. Particularly, all the compounds (6a-g and
9a-g) showed the ability to inhibit the RT containing the V106A mutation, with exception of derivatives 6e and 9a.
More interestingly, 6-chloro-1-[(3-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one derivative (6f), characterized by
the best antiretroviral profile, was also the best RT mutant inhibitor, active against all the single amino acid mutations with the
exception of Y188L mutation.
Table 3. HIV-1 RT inhibitory activity of compounds 6 (a-g) and 9 (a-g) against RT mutant enzyme carrying single amino acid
substitutions
L100I
IC50 (µM) a
K103N
IC50 (µM) a
V106A
IC50 (µM) a
E138K
IC50 (µM) a
Y181C
IC50 (µM) a
Y188L
IC50 (µM) a
G190A
IC50 (µM) a
Compound
6a
6b
90 71
7.13 5.0
29 19
92 69
≥64
>371
>335
>335
>335
9.38 1.6
1.8 0.1
6.6 0.09
15 0.0
>356
98 50
12 1.8
64 57
≥285
≥273
>335
>335
>335
>371
>335
>335
>335
250 73
238 27
260 30
>335
6c
6d
6e
>356
>356
>356
>356
>356
6f
6g
3.00 1.32
55 30
>356
214 3
≥285
180 18
59 8
65 5
311 44
0.27 0.0
14 0.0
105 7
280 29
>356
>323
>323
231 68
89 11
119 11
>342
0.63 0.0
188 0.0
0.90 0.18
6.6 0.3
>356
137 42
45 13
69 20
44 25
74 17
95 38
7.6 2.7
63 12
>356
≥323
>323
108 46
49 14
88 28
160 50
0.03 0.0
1.13 0.0
98 18
183 20
>356
176 24
74 24
107 3
32 8
>356
280 31
>356
>323
132 56
≥310
>356
9a
9b
280 1.3
258 9.6
69 6.8
54 13
90 1.6
89 0.0
0.057 0.00
>375
9c
9d
9e
>323
227 10
165 10
219 14
249 56
1.2 0.00
>375
9f
9g
80 2
179 22
0.03 0.00
>375
Efavirenz
Nevirapine
0.1 0.0
>375
a Compound concentration IC50 (µM) required to inhibit by 50% the RT activity of the indicated strain

Conclusion
In this study, two novel series of benzimidazolone analogues 6(a-g) and 9(a-g), acting as RT inhibitors, have been designed and
synthesized. The linker moiety between the phenyl ring and the sulfonyl group was modified and new small lipophilic groups on the
benzyl sulfonyl pendant were inserted. While the introduction of an ethylene bridge was detrimental for the enzymatic activity 9(a-g),
promising compounds have been obtained when a methylene bridge has been inserted 6(a-g). The best results were obtained for the 6-
chloro-1-[(3-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6f) with an IC50 value of 0.08 µM, twice as active against
HIV-1 than nevirapine (EC50 = 0.18 µM). In addition, compound 6f displayed also the best RT mutant inhibition profile. Moreover,
molecular docking studies were performed on both the two series of compounds.
Experimental section
Chemistry
All starting materials and reagents commercially available (Sigma-Aldrich Milan, Italy and Alfa Aesar, Karlsruhe, Germany) were
used without further purification. Melting points were determined on a BUCHI Melting Point B-545 apparatus and are uncorrected.
Elemental analyses (C, H, N) were carried out on a C. Erba Model 1106 Elemental Analyser and the results were within 0.4% of the
theoretical values and purity of tested compounds was >95%. Merck silica gel 60 F254 plates were used for TLC; Flash Chromatography
(FC) was performed on Biotage SP1 EXP. 1H NMR spectra were measured with a Varian Gemini-300 spectrometer in CDCl3 with TMS
as internal standard or in DMSO-d6. Coupling constants (J) are reported in hertz and chemical shifts are expressed in δ (ppm). All
exchangeable protons were confirmed by addition of deuterium oxide (D2O).
General procedures for the synthesis of 2-phenylethanesulfonyl chlorides (3a-g)
The appropriate phenylethyl bromide 2 (a-g) (1 mmol) was dissolved in MeCN (2mL) and KSAc (114 mg, 1 mmol) was added in an
ice-bath. After stirring for 1h, 1mL of HCl conc. was added followed by the slow addition of NaClO2 (360 mg, 4 mmol). The obtained
mixture was stirred for 40 minutes and then the reaction was quenched adding water (3 mL). The resulting solution was evaporated in
vacuum to remove the solvent, extracted with EtOAc (3x10 mL) and dried over anhydrous Na2SO4. Finally, the solution was
concentrated in vacuum to obtain the desired compounds 3 (a-g).
2-Phenylethanesulfonyl chloride (3a)
Yield 63%; yellowish oil. 1H NMR (CDCl3): 2.62-2.68 (m, 2H, CH2), 2.82-2.88 (m, 2H, CH2), 7.14-7.27 (m, 5H, ArH). Anal. Calcd
for C8H9ClO2S: C: 46.95; H: 4.43; O: 15.63. Found: C: 47.01; H: 4.35; O: 15.78.
2-(2-Chlorophenyl)ethanesulfonyl chloride (3b)
Yield 63%; yellowish oil. 1H NMR (CDCl3): 3.27-3.32 (m, 2H, CH2), 3.57-3.62 (m, 2H, CH2), 7.20-7.45 (m, 4H, ArH). Anal. Calcd
for C8H8Cl2O2S: C: 40.18; H: 3.37; O: 13.38. Found: C: 39.99; H: 3.48; O: 13.50.
2-(3-Chlorophenyl)ethanesulfonyl chloride (3c)
1
Yield 60%; yellowish oil. H NMR (CDCl3): 3.27-3.34 (m. 2H, CH2), 3.87-3.99 (m, 2H, CH2), 7.12-7.15 (m, 1H, ArH), 7.24-7.30
(m, 3H, ArH). Anal. Calcd for C8H8Cl2O2S: C: 40.18; H: 3.37; O: 13.38. Found: C: 40.22; H: 3.25; O: 13.49.
2-(4-Chlorophenyl)ethanesulfonyl chloride (3d)
Yield 55%; yellowish oil. 1H NMR (CDCl3): 2.60-2.66 (m. 2H, CH2), 2.81-2.86 (m, 2H, CH2), 7.21 (d, J= 8.8, 2H, ArH), 7.28 (d, J=
8.8, 2H, ArH). Anal. Calcd for C8H8Cl2O2S: C: 40.18; H: 3.37; O: 13.38. Found: C: 40.30; H: 3.19, O: 13.33.
2-(2-Methylphenyl)ethanesulfonyl chloride (3e)
Yield 68%; yellowish oil. 1H NMR (CDCl3): 2.29 (s, 3H, CH3), 3.10-3.23 (m. 2H, CH2), 3.47-3.52 (m, 2H, CH2), 7.07-7.29 (m, 4H,
ArH). Anal. Calcd for C9H11ClO2S: C: 49.43; H: 5.07; O: 14.63. Found: C: 49.55; H: 4.96, O: 14.73.
2-(3-Methylphenyl)ethanesulfonyl chloride (3f)
Yield 54%; yellowish oil. 1H NMR (CDCl3): 2.24 (s, 3H, CH3), 2.61-2.64 (m. 2H, CH2), 2.77-2.83 (m, 2H, CH2), 6.97 (t, J= 7.6, 3H,
ArH), 7.28 (t, J= 7.6, 1H, ArH). Anal. Calcd for C9H11ClO2S: C: 49.43; H: 5.07; O: 14.63. Found: C: 49.33; H: 5.00, O: 14.88.
2-(4-Methylphenyl)ethanesulfonyl chloride (3g)
1
Yield 55%; yellowish oil. H NMR (CDCl3): 2.36 (s, 3H, CH3), 3.08-3.13 (m. 2H, CH2), 3.51-3.57 (m, 2H, CH2), 7.01 (d, J= 7.7,
2H, ArH), 7.12-7.21 (m, 2H, ArH). Anal. Calcd for C9H11ClO2S: C: 49.43; H: 5.07; O: 14.63. Found: C: 49.59; H: 5.11, O: 14.42.
General procedures for the synthesis of N-(5-chloro-2-nitrophenyl)-1-(substituted-phenyl)methanesulfonamides 4 (a-g) and N-(5-
chloro-2-nitrophenyl)-2-(substituted-phenyl)ethanesulfonamides 7 (a-g)
5-Chloro-2-nitroaniline (173 mg, 1 mmol) was dissolved in DMF (3 mL) and dry sodium hydride (120 mg, 5 mmol) was added. The
obtained mixture was stirred for 1 min and the suitable substituted benzensulfonyl chloride 1 (a-g) or phenylethanesulfonyl chloride 3
(a-g) (1,5 mmol) was added in several portions. The reaction was quenched after 2-6 h through the addition of a saturated NaHCO3
aqueous solution (5 mL). The mixture was extracted with EtOAc (3 x 10 mL) and dried over Na2SO4. After removal of the solvent

under reduced pressure, the residue was purified by flash chromatography using cyclohexane/AcOEt (80:20) as eluent and crystallized
from Et2O.
N-(5-chloro-2-nitrophenyl)-1-phenylmethanesulfonamide (4a)
Mp: 125-127 °C, yield 42 %. 1H NMR (CDCl3): 4.50 (s, 2H, CH2), 7.08 (dd, J= 8.8, J= 2.2, 1H, ArH), 7.21-7.37 (m, 5H, ArH), 7.59
(d, 1H, J= 2.2, ArH), 8.10 (d, J= 8.8, 1H, ArH), 9.68 (bs, 1H, NH). Anal. Calcd for C13H11ClN2O4S: C: 47.79; H: 3.39; N: 8.57. Found:
C: 47.88; H: 3.21; N: 8.78.
N-(5-chloro-2-nitrophenyl)-1-(2-chlorophenyl)methanesulfonamide (4b)
Mp: 118-120 °C, yield 55%. 1H NMR (CDCl3): 4.76 (s, 2H, CH2), 7.05 (dd, J= 9.0, J= 2.2, 1H, ArH), 7.21-7.33 (m, 3H, ArH), 7.57
(dd, J= 8.0, J= 2.2, 1H, ArH), 7.64 (d, 1H, J= 2.2, ArH), 8.12 (d, J= 9.0, 1H, ArH), 9.90 (bs, 1H, NH). Anal. Calcd for C13H10Cl2N2O4S:
C: 43.23; H: 2.79; N: 7.76. Found: C: 43.13; H: 2.89; N: 7.66.
N-(5-chloro-2-nitrophenyl)-1-(3-chlorophenyl)methanesulfonamide (4c)
1
Mp: 175-177 °C, yield 63 %. H NMR (CDCl3): 4.47 (s, 2H, CH2), 7.12 (d, J= 9.0, J= 2.1, 1H, ArH), 7.16-7.31 (m, 4H, ArH), 7.66
(d, 1H, J= 2.1, ArH), 8.14 (d, J= 9.0, 1H, ArH), 9.73 (bs, 1H, NH). Anal. Calcd for C13H10Cl2N2O4S: C: 43.23; H: 2.79; N: 7.76. Found:
C: 43.11; H: 2.95; N: 7.63.
N-(5-chloro-2-nitrophenyl)-1-(4-chlorophenyl)methanesulfonamide (4d)
1
Mp: 137-139 °C, yield 39 %. H NMR (CDCl3): 4.47 (s, 2H, CH2), 7.12 (dd, J= 8.7, J= 2.4, 1H, ArH), 7.18-7.29 (d, J= 8.8, 2H,
ArH), 7.56 (d, 1H, J= 2.4, ArH), 8.12 (d, J= 8.7, 1H, ArH), 9.72 (bs, 1H, NH). Anal. Calcd for C13H10Cl2N2O4S: C: 43.23; H: 2.79; N:
7.76. Found: C: 43.13; H: 2.89; N: 7.86.
N-(5-chloro-2-nitrophenyl)-1-(2-methylphenyl)methanesulfonamide (4e)
1
Mp: 134-136°C, yield 81 %. H NMR (CDCl3): 2.32 (s, 3H, CH3), 4.95 (s, 2H, CH2), 6.98-7.18 (m, 5H, ArH), 7.55 (s, 1H, ArH),
8.00 (d, J= 8.9, 1H, ArH), 9.41 (bs, 1H, NH). Anal. Calcd for C14H13ClN2O4S: C: 49.34; H: 3.85; N: 8.22. Found: C: 49.58; H: 3.69; N:
8.36.
N-(5-chloro-2-nitrophenyl)-1-(3-methylphenyl)methanesulfonamide (4f)
1
Mp: 141-143 °C, yield 48 %. H NMR (CDCl3): 2.25 (s, 3H, CH3), 4.45 (s, 2H, CH2), 7.00-7.25 (m, 5H, ArH), 7.60 (d, 1H, J= 2.4,
ArH), 8.08 (d, J= 9.4, 1H, ArH), 9.66 (bs, 1H, NH). Anal. Calcd for C14H13ClN2O4S: C: 49.34; H: 3.85; N: 8.22. Found: C: 49.44; H:
3.91; N: 8.10.
N-(5-chloro-2-nitrophenyl)-1-(4-methylphenyl)methanesulfonamide (4g)
1
Mp: 125-127 °C, yield 53 %. H NMR (CDCl3): 2.30 (s, 3H, CH3), 4.46 (s, 2H, CH2), 7.03-7.12 (m, 5H, ArH), 7.48 (s, 1H, ArH),
8.08 (d, J= 9.0, 1H, ArH), 9.68 (bs, 1H, NH). Anal. Calcd for C14H13ClN2O4S: C: 49.34; H: 3.85; N: 8.22. Found: C: 49.36; H: 3.72; N:
8.37.
N-(5-chloro-2-nitrophenyl)-2-phenylethanesulfonamide (7a)
1
Yield 45%; yellowish oil. H NMR (CDCl3): 3.00-3.08 (m, 2H, CH2), 3.40-3.51 (m, 2H, CH2), 7.09 (d, J= 8.8, 1H, ArH), 7.21-7.37
(m, 5H, ArH), 9.78 (bs, 1H, NH). Anal. Calcd for C14H13ClN2O4S: C: 49.34; H: 3.85; N: 8.22. Found: C: 49.40; H: 3.99, N: 8.01.
N-(5-chloro-2-nitrophenyl)-2-(2-chlorophenyl)ethanesulfonamide (7b)
Yield 33%; yellowish oil. 1H NMR (CDCl3): 3.15-3.20 (m, 2H, CH2), 3.53-3.58 (m, 2H, CH2), 7.01-7.07 (m, 1H, ArH), 7.10 (s, 2H,
ArH), 7.15-7.20 (m, 2H, ArH), 7.88 (s, 1H, ArH), 8.18 (d, J= 9.3, 1H, ArH), 10.11 (bs, 1H, NH). Anal. Calcd for C14H12Cl2N2O4S: C:
44.81; H: 3.22; N: 7.47. Found: C: 44.91; H: 3.33; N: 7.20.
N-(5-chloro-2-nitrophenyl)-2-(3-chlorophenyl)ethanesulfonamide (7c)
1
Yield 51%, Mp: 170-172 °C. H NMR (CDCl3): 3.13-3.18 (m, 2H, CH2), 3.52-3.57 (m, 2H, CH2), 7.03-7.17 (m, 5H, ArH), 7.86 (s,
1H, ArH), 8.16 (d, J= 9.3, 1H, ArH), 9.72 (bs, 1H, NH). Anal. Calcd for C14H12Cl2N2O4S: C: 44.81; H: 3.22; N: 7.47. Found: C: 44.99;
H: 3.17; N: 7.59.
N-(5-chloro-2-nitrophenyl)-2-(4-chlorophenyl)ethanesulfonamide (7d)
Yield 52%; yellowish oil. 1H NMR (CDCl3): 3.15-3.19 (m, 2H, CH2), 3.53-3.60 (m, 2H, CH2), 7.00-7.22 (m, 5H, ArH), 7.88 (s, 1H,
ArH), 8.19 (d, J= 9.0, 1H, ArH), 9.73 (bs, 1H, NH). Anal. Calcd for C14H12Cl2N2O4S: C: 44.81; H: 3.22; N: 7.47. Found: C: 44.75; H:
3.36; N: 7.60.
N-(5-chloro-2-nitrophenyl)-2-(2-methylphenyl)ethanesulfonamide (7e)
1
Yield 37%, Mp: 192-194°C. H NMR (CDCl3): 2.36 (s, 3H, CH3), 2.58-2.64 (m, 2H, CH2), 2.84-2.95 (m, 2H, CH2), 7.04-7.18 (m,
6H, ArH), 8.20 (s, 1H, ArH), 9.65 (bs, 1H, NH). Anal. Calcd for C15H15ClN2O4S: C: 50.78; H: 4.26; N: 7.90. Found: C: 50.61; H: 4.13;
N: 8.01.
N-(5-chloro-2-nitrophenyl)-2-(3-methylphenyl)ethanesulfonamide (7f)

Yield 61%; yellowish oil. 1H NMR (CDCl3): 2.38 (s, 3H, CH3), 2.85-2.98 (m, 2H, CH2), 3.07-3.13 (m, 2H, CH2), 7.04-7.18 (m, 6H,
ArH), 8.22 (s, 1H, ArH), 11.04 (bs, 1H, NH). Anal. Calcd for C15H15ClN2O4S: C: 50.78; H: 4.26; N: 7.90. Found: C: 50.68; H: 4.09; N:
8.00.
N-(5-chloro-2-nitrophenyl)-2-(4-methylphenyl)ethanesulfonamide (7g)
Yield 40%; yellowish oil.1H NMR (CDCl3): 2.19 (s, 3H, CH3), 2.83-2.89 (m, 2H, CH2), 3.16-3.23 (m, 2H, CH2), 6.60-6.75 (m, 5H,
ArH), 7.07-7.16 (m, 2H, ArH). Anal. Calcd for C15H15ClN2O4S: C: 50.78; H: 4.26; N: 7.90. Found: C: 50.79; H: 4.35; N: 7.92.
General procedures for the synthesis of N-(2-amino-5-chlorophenyl)-1-(substituted-phenyl)methanesulfonamides 5(a-g) and N-(2-
amino-5-chlorophenyl)-2-(substituted-phenyl)ethanesulfonamides 8(a-g)
The appropriate N-(5-chloro-2-nitrophenyl)-1-phenylmethanesulfonamide 4(a-g) (1mmol) or N-(5-chloro-2-nitrophenyl)-2-
phenylethanesulfonamide 7(a-g) (1 mmol) was dissolved in HCl conc. (5 mL) and EtOH (7 mL), then zinc dust (2.18 g, 33 mmol) was
added in several portions at 0°C. When the addition was completed the reaction was refluxed for 1h at 100°C. The resulting mixture
was cooled at room temperature, made alkaline with NaOH 5N aqueous solution and extracted with EtOAc (3 x 10 mL). The organic
phase was dried over Na2SO4 and evaporated to remove the solvent. The residue was crystallized from EtOH or purified by column
flash chromatography using a mixture of cyclohexane/AcOEt (70:30) as eluent.
N-(2-amino-5-chlorophenyl)-1-phenylmethanesulfonamide (5a)
1
Mp: 164-166 °C, yield 93 %. H NMR (DMSO-d6): 4.31 (s, 2H, CH2), 5.15 (bs, 2H, NH2), 6.67 (d, J= 8.2, 1H, ArH), 6.88 (dd, J=
8.2, J= 2.3, 1H, ArH), 7.03 (d, 1H, J= 2.3, ArH), 7.32 (mc, 5H, ArH). Anal. Calcd for C13H13ClN2O2S: C: 52.61; H: 4.42; N: 9.44.
Found: C: 52.81; H: 4.58; N: 9.39.
N-(2-amino-5-chlorophenyl)-1-(2-chlorophenyl)methanesulfonamide (5b)
1
Mp: 145-147 °C, yield 87 %. H NMR (DMSO-d6):4.54 (s, 2H, CH2), 5.29 (bs, 2H, NH2), 6.72 (d, J= 8.5, 1H, ArH), 6.97 (dd, J=
8.5, J= 2.6, 1H, ArH), 7.03 (d, s, 1H, J= 2.6, ArH), 7.35-7.49 (m, 4H, ArH). Anal. Calcd for C13H12Cl2N2O2S: C: 47.14; H: 3.65; N:
8.46. Found: C: 47.25; H: 3.75; N: 8.36.
N-(2-amino-5-chlorophenyl)-1-(3-chlorophenyl)methanesulfonamide (5c)
1
Mp: 167-169 °C, yield 91 %. H NMR (DMSO-d6): 4.43 (s, 2H, CH2), 5.24 (bs, 2H, NH2), 6.71 (d, J= 8.7, 1H, ArH), 6.97 (dd, J=
8.7, J= 2.3, 1H, ArH), 7.03 (d, 1H, J= 2.3, ArH), 7.29-7.41 (m, 4H, ArH). Anal. Calcd for C13H12Cl2N2O2S: C: 47.14; H: 3.65; N: 8.46.
Found: C: 47.24; H: 3.55; N: 8.55.
N-(2-amino-5-chlorophenyl)-1-(4-chlorophenyl)methanesulfonamide (5d)
1
Mp: 156-158 °C, yield 94 %. H NMR (DMSO-d6): 4.38 (s, 2H, CH2), 5.20 (bs, 2H, NH2), 6.69 (d, J= 8.5, 1H, ArH), 6.93 (dd, J=
8.5, J= 2.6, 1H, ArH), 7.01 (d, 1H, J= 2.6, ArH), 7.33-7.42 (m, 4H, ArH). Anal. Calcd for C13H12Cl2N2O2S: C: 47.14; H: 3.65; N: 8.46.
Found: C: 47.28; H: 3.41; N: 8.63.
N-(2-amino-5-chlorophenyl)-1-(2-methylphenyl)methanesulfonamide (5e)
1
Mp: 155-157°C, yield 91 %. H NMR (DMSO-d6): 2.33 (s, 3H, CH3), 4.24 (s, 2H, CH2), 5.18 (bs, 2H, NH2), 6.75 (d, J=8.4, 1H,
ArH), 6.70 (d, J=8.4, 1H, ArH), 7.01 (s, 1H, ArH), 7.35-7.52 (m, 4H, ArH). Anal. Calcd for C14H15ClN2O2S: C: 54.10; H: 4.86; N: 9.01.
Found: C: 54.27; H: 4.81; N: 8.92.
N-(2-amino-5-chlorophenyl)-1-(3-methylphenyl)methanesulfonamide (5f)
1
Mp:163-165°C, yield 88 %. H NMR (DMSO-d6): 2.29 (s, 3H, CH3), 4.20 (s, 2H, CH2), 5.23 (bs, 2H, NH2), 6.75 (d, J= 8.0, 1H,
ArH), 6.72 (d, J= 8.0, 1H, ArH), 7.00 (s, 1H, ArH), 7.27-7.41 (m, 4H, ArH). Anal. Calcd for C14H15ClN2O2S: C: 54.10; H: 4.86; N:
9.01. Found: C: 54.30; H: 4.65; N: 9.09.
N-(2-amino-5-chlorophenyl)-1-(4-methylphenyl)methanesulfonamide (5g)
1
Mp: 168-170 °C, yield 94 %. H NMR (DMSO-d6): 2.27 (s, 3H, CH3), 4.29 (s, 2H, CH2), 5.19 (bs, 2H, NH2), 6.68 (d, J= 8.5, 1H,
ArH), 6.92 (dd, J= 8.5, J= 2.2, 1H, ArH), 6.97 (d, J= 2.2, 1H, ArH), 7.13 (d, J= 8.5, 2H, ArH), 7.20 (d, J= 8.5, 2H, ArH). Anal. Calcd
for C14H15ClN2O2S: C: 54.10; H: 4.86; N: 9.01. Found: C: 54.20; H: 4.98; N: 9.11.
N-(2-amino-5-chlorophenyl)-2-phenylethanesulfonamide (8a)
Yield 55%, Mp: 134-136°C. 1H NMR (DMSO-d6): 2.97-3.00 (m, 2H, CH2), 3.23-3.25 (m, 2H, CH2), 5.32 (bs, 2H, NH2), 6.72 (d, J=
8.8, 1H, ArH), 6.99 (d, J= 8.8, 1H, ArH), 7.09 (s, 1H, ArH), 7.20-7.26 (m, 5H, ArH). Anal. Calcd for C14H15ClN2O2S: C: 54.10; H:
4.86; N: 9.01. Found: C: 54.28; H: 4.99; N: 9.27.
N-(2-amino-5-chlorophenyl)-2-(2-chlorophenyl)ethanesulfonamide (8b)
1
Yield 50%; yellowish oil. H NMR (DMSO-d6): 3.14-3.20 (m, 2H, CH2 ), 3.38-3.43 (m, 2H, CH2), 4.08 (bs, 2H, NH2), 6.74 (d, J=
8.3, 1H, ArH), 7.02 (s, 1H, ArH), 7.11-7.16 (m, 2H, ArH), 7.22-7.29 (m, 3H, ArH), 10.03 (bs, 1H, NH). Anal. Calcd for
C14H14Cl2N2O2S: C: 48.70; H: 4.09; N: 8.11. Found: C: 48.98; H: 3.91; N: 7.95.
N-(2-amino-5-chlorophenyl)-2-(3-chlorophenyl)ethanesulfonamide (8c)

1
Mp: 150-152 °C, yield 44%. H NMR (CDCl3): 3.13-3.19 (m, 2H, CH2 ), 3.36-3.42 (m, 2H, CH2), 4.07 (bs, 2H, NH2), 5.89 (s, 1H,
ArH), 6.72 (d, J= 9.3, 1H, ArH),7.01 (s, 1H, ArH), 7.05-7.12 (m, 2H, ArH), 7.23-7.27 (m, 2H, ArH). Anal. Calcd for C14H14Cl2N2O2S:
C: 48.70; H: 4.09; N: 8.11. Found: C: 48.88; H: 4.02; N: 8.36.
N-(2-amino-5-chlorophenyl)-2-(4-chlorophenyl)ethanesulfonamide (8d)
1
Yield 47%, yellowish oil. H NMR (CDCl3): 2.82-2.87 (m, 2H, CH2 ), 3.15-3.18 (m, 2H, CH2), 3.40 (bs, 2H, NH2), 6.59-6.74 (m,
4H, ArH), 7.15 (d, J= 8.8, 2H, ArH), 7.29 (s, 1H, ArH). Anal. Calcd for C14H14Cl2N2O2S: C: 48.70; H: 4.09; N: 8.11. Found: C: 48.91;
H: 4.11; N: 7.93.
N-(2-amino-5-chlorophenyl)-2-(2-methylphenyl)ethanesulfonamide (8e)
Yield 43%, yellowish oil. 1H NMR (CDCl3): 2.86 (s, 3H, CH3), 2.88-2.94 (m, 2H, CH2 ), 3.01-3.08 (m, 2H, CH2 ), 4.00 (bs, 2H, NH2
), 6.90 (s, 1H, ArH), 7.19-7.31 (m, 4H, ArH), 7.49 (s, 1H, ArH), 8.08 (s, 1H, ArH). Anal. Calcd for C15H17ClN2O2S: C: 55.46; H: 5.28;
N: 8.62. Found: C: 55.58; H: 5.40; N: 8.37.
N-(2-amino-5-chlorophenyl)-2-(3-methylphenyl)ethanesulfonamide (8f)
1
Yield 47%, yellowish oil. H NMR (CDCl3): 2.35 (s, 3H, CH3), 3.23-3.29 (m, 2H, CH2 ), 3.37-3.45 (m, 2H, CH2), 4.09 (bs, 2H,
NH2), 5.87 (s, 1H, ArH), 6.73 (d, J= 8.2, 1H, ArH), 7.02-7.18 (m, 4H, ArH), 7.27 (s, 1H, ArH).Anal. Calcd for C15H17ClN2O2S: C:
55.46; H: 5.28; N: 8.62. Found: C: 55.30; H: 4.99; N: 8.94.
N-(2-amino-5-chlorophenyl)-2-(4-methylphenyl)ethanesulfonamide (8g)
Yield 73%, yellowish oil. 1H NMR (CDCl3):2.19 (s, 3H, CH3), 2.83-2.90 (m, 2H, CH2 ), 3.16-3.20 (m, 2H, CH2), 3.38 (bs, 2H, NH2),
6.60-6.75 (m, 5H, ArH), 7.15-7.19 (m, 2H, ArH).Anal. Calcd for C15H17ClN2O2S: C: 55.46; H: 5.28; N: 8.62. Found: C: 55.36; H: 5.37;
N: 8.77.
General procedures for the synthesis of 6-chloro-1-[(substituted-benzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-ones 6 (a-g) and
6-chloro-1-{[2-(substituted-phenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-ones 9(a-g)
To a solution of the appropriate N-(2-amino-5-chlorophenyl)-1-phenylmethanesulfonamide 5 (a-g) or N-(2-amino-5-chlorophenyl)-
2-phenylethanesulfonamide 8 (a-g) (1 mmol) in 12N HCl (1 mL) and H2O (2 mL), an excess of a 20% toluene solution of phosgene
(3.8 mL, 36 mmol) was added and the resulting mixture was heated for 4h at 100°C. After cooling, the reaction mixture was alkalized
with NaOH 5N and extracted with EtOAc (3 x 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced
pressure. The residue was crystallized from Et2O to obtain the desired compounds 6(a-g) and 9(a-g).
1-(benzylsulfonyl)-6-chloro-1,3-dihydro-2H-benzimidazol-2-one (6a)
Mp: 188-190 °C, yield 53%. 1H NMR (DMSO-d6): 4.98 (s, 2H, CH2), 6.82 (s, 1H, ArH), 6.88 (dd, J= 8.5, J= 2.1, 1H, ArH), 7.01 (d,
1H, J= 2.1, ArH), 7.24-7.26 (m, 5H, ArH), 11.77 (bs, 1H, NH). Anal. Calcd for C14H11ClN2O3S: C: 52.10; H: 3.44; N: 8.68. Found: C:
52.22; H: 3.59; N: 8.75.
6-chloro-1-[(2-chlorobenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6b)
1
Mp: 195-197 °C, yield 37%. H NMR (DMSO-d6): 5.22 (s, 2H, CH2), 6.97 (s, 1H, ArH), 7.01-7.14 (m, 2H, ArH), 7.35 (mc, 3H,
ArH), 7.56 (mc, 1H, ArH), 11.65 (bs, 1H, NH). Anal. Calcd for C14H10Cl2N2O3S: C: 47.07; H: 2.82; N: 7.84. Found: C: 47.01; H: 2.92;
N: 7.94.
6-chloro-1-[(3-chlorobenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6c)
Mp: 212-214 °C, yield 67 %. 1H NMR (DMSO-d6): 5.11 (s, 2H, CH2), 7.00 (d, J= 8.8, 1H, ArH), 7.12-7.14 (m, 2H, ArH), 7.26-7.34
(m, 4H, ArH), 11.85 (bs, 1H, NH). Anal. Calcd for C14H10Cl2N2O3S: C: 47.07; H: 2.82; N: 7.84. Found: C: 47.17; H: 2.92; N: 7.94.
6-chloro-1-[(4-chlorobenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6d)
Mp: 186-188 °C, yield 62 %. 1H NMR (DMSO-d6): 4.99 (s, 2H, CH2), 6.78 (d, J= 8.5, 1H, ArH), 6.86 (dd, J= 8.5, J= 2.1, 1H, ArH),
7.00 (s, 1H, J= 2.1, ArH), 7.26-7.34 (m, 4H, ArH) 10.78 (bs, 1H, NH). Anal. Calcd for C14H10Cl2N2O3S: C: 47.07; H: 2.82; N: 7.84.
Found: C: 47.23; H: 3.06; N: 7.59.
6-chloro-1-[(2-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6e)
1
Mp: 218-220°C, yield 44%. H NMR (DMSO-d6): 2.32 (s, 3H, CH3), 4.98 (s, 2H, CH2), 6.75-7.00 (m, 3H, ArH), 7.16-7.21 (m, 4H,
ArH), 11.16 (bs, 1H, NH). Anal. Calcd for C15H13ClN2O3S: C: 53.49; H: 3.89; N: 8.32. Found: C: 53.76; H: 3.99; N: 8.54.
6-chloro-1-[(3-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6f)
Mp: 193-195 °C, yield 58 %. 1H NMR (DMSO-d6): 2.16 (s, 3H, CH3 ), 4.91 (s, 2H, CH2 ), 6.80 (d, 1H, J= 8.0, ArH), 6.86- 6.92 (m,
2H, ArH), 7.01-7.13 (m, 4H, ArH), 10.77 (bs, 1H, NH). Anal. Calcd for C15H13ClN2O3S: C: 53.49; H: 3.89; N: 8.32. Found: C: 53.59;
H: 4.10; N: 8.44.
6-chloro-1-[(4-methylbenzyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (6g)
1
Mp: 188-190 °C, yield 43 %. H NMR (DMSO-d6): 2.20 (s, 3H, CH3 ), 4.93 (s, 2H, CH2 ), 6.81 (d, J= 8.0, 1H, ArH), 6.92 (dd, J=
8.5, J= 2.1, 1H, ArH),), 7.01 (d, J= 2.1, 1H, ArH), 7.03 (t, J= 8.0, 3H, ArH), 7.13 (d, J= 8.5, 2H, ArH), 10.77 (bs, 1H, NH). Anal. Calcd
for C15H13ClN2O3S: C: 53.49; H: 3.89; N: 8.32. Found: C: 53.27; H: 4.02; N: 8.58.

6-Chloro-1-[(2-phenylethyl)sulfonyl]-1,3-dihydro-2H-benzimidazol-2-one (9a)
Yield 61%, Mp: 183-185°C. 1H NMR (DMSO-d6): 3.02-3.06 (m, 2H, CH2), 4.08-4.13 (m, 2H, CH2), 6.95 (d, J= 8.8, 1H, ArH), 7.10-
7.18 (m, 6H, ArH), 7.46 (s, 1H, ArH), 9.33 (bs, 1H, NH). Anal. Calcd for C15H13ClN2O3S: C: 53.49; H: 3.89; N: 8.32. Found: C: 53.59;
H: 3.79; N: 8.42.
6-Chloro-1-{[2-(2-chlorophenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9b)
Yield 81%, Mp: 190-192°C. 1H NMR (DMSO-d6): 2.96-3.02 (m, 2H, CH2), 3.84-3.89 (m, 2H, CH2), 6.79 (d, J= 8.2, 1H, ArH), 6.87-
6.94 (m, 4H, ArH), 7.23-7.37 (m, 2H, ArH), 10.81 (bs, 1H, NH). Anal. Calcd for C15H12Cl2N2O3S: C: 48.53; H: 3.26; N: 7.55. Found:
C: 48.78; H: 2.99; N: 7.86.
6-Chloro-1-{[2-(3-chlorophenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9c)
1
Yield 43%, Mp: 230-232°C. H NMR (DMSO-d6): 3.06-3.11 (m, 2H, CH2 ), 4.14-4.18 (m, 2H, CH2), 6.90-6.97 (m, 3H, ArH), 7.15-
7.17 (m, 2H, ArH), 7.25 (s, 1H, ArH), 7.43 (s, 1H, ArH), 10.77 (bs, 1H, NH). Anal. Calcd for C15H12Cl2N2O3S: C: 48.53; H: 3.26; N:
7.55. Found: C: 48.69; H: 3.01; N: 7.64.
6-Chloro-1-{[2-(4-chlorophenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9d)
1
Yield 54%, Mp: 295-297°C. H NMR (DMSO-d6): 3.04-3.09 (m, 2H, CH2 ), 4.11-4.16 (m, 2H, CH2), 6.98 (d, J= 8.8, 2H, ArH),
7.13-7.20 (m, 4H, ArH), 7.49 (s, 1H, ArH), 10.12 (bs, 1H, NH). Anal. Calcd for C15H12Cl2N2O3S: C: 48.53; H: 3.26; N: 7.55. Found: C:
48.31; H: 3.43; N: 7.79.
6-Chloro-1-{[2-(2-methylphenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9e)
Yield 41%, Mp: 118-120°C.1H NMR (DMSO-d6): 2.45 (s, 3H, CH3), 3.27-3.39 (m, 2H, CH2 ), 4.31-4.36 (m, 2H, CH2), 7.12-7.19
(m, 2H, ArH), 7.29-7.67 (m, 5H, ArH), 10.97 (bs, 1H, NH).Anal. Calcd for C16H15ClN2O3S: C: 54.78; H: 4.31; N: 7.99. Found: C:
54.88; H: 4.60; N: 7.71.
6-Chloro-1-{[2-(3-methylphenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9f)
Yield 91%, Mp: 117-119°C.1H NMR (DMSO-d6): 2.22 (s, 3H, CH3), 3.07-3.12 (m, 2H, CH2 ), 4.04-4.09 (m, 2H, CH2), 6.84-6.91
(m, 2H, ArH), 7.01-7.35 (m, 5H, ArH), 10.74 (bs, 1H, NH).Anal. Calcd for C16H15ClN2O3S: C: 54.78; H: 4.31; N: 7.99. Found: C:
54.61; H: 4.24; N: 8.15.
6-Chloro-1-{[2-(4-methylphenyl)ethyl]sulfonyl}-1,3-dihydro-2H-benzimidazol-2-one (9g)
Yield 45%, Mp: 125-127°C.1H NMR (CDCl3): 2.20 (s, 3H, CH3), 3.95-4.04 (m, 2H, CH2 ), 4.51-4.58 (m, 2H, CH2), 6.62-6.68 (m,
1H, ArH), 6.92-7.01 (m, 2H, ArH), 7.09-7.26 (m, 4H, ArH), 9.29 (bs, 1H, NH). Anal. Calcd for C16H15ClN2O3S: C: 54.78; H: 4.31; N:
7.99. Found: C: 54.67; H: 4.49; N: 8.01.
Docking studies
Preparation of RT protein. The reverse transcriptase protein was prepared using Autodock Vina.21 The protein was retrieved from
Protein Data Bank (PDB: 3DLG). The water molecules solved by X-ray crystallography were removed from the protein while the
missing hydrogens were added to the pattern.
Preparation of the ligands. 3D structure of each ligand was constructed using Discovery Studio2.5.5. They were minimized using
CHARMm forcefield followed by Smart Minimizer algorithm. 1000 Steps of Steepest Descent with a root mean square (RMS) gradient
tolerance of 3 were performed, followed by Conjugate Gradient minimization, until the RMS gradient for potential energy was less than
⁄mol⁄Å. AutoDockTools 1.5.4 was used to
0.05 kcal
additionally process the designed 3D structures by assigning the correct AutoDock
atom types to their atoms, adding Gasteiger charges, merging non-polar hydrogens, detecting aromatic carbons if any, and setting up the
'torsion tree'. The same was done with GW564511 for re-docking procedure.
Grid box and Automated Docking. A grid box with a dimension of 20 x 20 x 20 size with spacing of 0.415 was defined around the
crystallized ligand to cover the entire non-nucleoside binding site and accommodate ligands to move freely. Then automated docking
studies were carried out using the empirical free energy function and the Lamarckian Genetic Algorithm (LGA). A standard protocol
was applied, with an initial population of 150 randomly placed individuals and a maximum number of 2.5 ×106 energy evaluations, a
maximum number of 27,000 generations, a mutation rate of 0.02, a crossover rate of 0.80 and an elitism value of 1. A total of 50
independent docking runs were carried out for each compound and the parameters were set using ADT. Structures differing by less than
2.0 Å in positional root-mean-square deviation (RMSD) were clustered together and the most favourable free binding energy complex
structure was selected.
Docking Protocol validation. The validation of the docking protocol was performed by docking the native co-crystallized ligand
GW564511 into RT binding site. The comparison of docking results with the co-crystallized form showed success rates with the docked
ligand strictly superimposed with the crystallized conformation with RMSD = 0.50 Å indicating that the used scoring function is
successful. These values were small enough and supported the hypothesis that experimental binding modes could be reproduced with
accuracy using this protocol.
All figures were generated using the PyMOL software. 24

Biological assays
In vitro anti-HIV assay. Evaluation of the antiviral activity of the compounds against HIV in MT-4 cells was performed using the
MTT assay as previously described. 25 Stock solutions (10 x final concentration) of test compounds were added in 25 µl volumes to two
series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each
experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek
3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell samples were included as controls. HIV
stock (50 µl) at 100-300 CCID50 (50 % cell culture infectious doses) or culture medium was added to either the infected or mock-
infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in order
to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 minutes at 220 g and the
supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/ml and 50 µl volumes were transferred to the microtiter
tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically using the MTT
assay. The MTT assay is based on the reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue-purple formazan that can be
measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000,
Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median absorbance value of three wells. The 50%
cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-
infected control sample by 50%. The concentration achieving 50% protection against the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
Reverse transcriptase assay. Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as described by. 26 The RT assay
is performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by the manufacturer. The
assay is based on the dsDNA quantitation reagent PicoGreen. This reagent shows a pronounced increase in fluorescence signal upon
binding to dsDNA or RNA-DNA heteroduplexes. Single-stranded nucleic acids generate only minor fluorescence signal enhancement
when a sufficiently high dye:base pair ratio is applied. 27 This condition is met in the assay. A poly(rA) template of approximately 350
bases long, and an oligo(dT)16 primer, are annealed in a molar ratio of 1:1.2 (60 min. at room temperature). Fifty-two ng of the
RNA/DNA is brought into each well of a 96-well plate in a volume of 20 µl polymerization buffer (60 mMTris-HCl, 60 mMKCl, 8 mM
MgCl2, 13 mM DTT, 100 µM dTTP, pH 8.1). Five µl of RT enzyme solution, diluted to a suitable concentration in enzyme dilution
buffer (50 mMTris-HCl, 20% glycerol, 2 mM DTT, pH 7.6), is added. The reactions are incubated at 25°C for 40 minutes and then
stopped by the addition of EDTA (15 mM fc). Heteroduplexes are then detected by addition of PicoGreen. Signals are read using an
excitation wavelength of 490 nm and emission detection at 523 nm using a spectrofluorometer (Safire 2, Tecan). To test the activity of
compounds against RT, 1 µl of compound in DMSO is added to each well before the addition of RT enzyme solution. Control wells
without compound contain the same amount of DMSO. Results are expressed as relative fluorescence i.e. the fluorescence signal of the
reaction mix with compound divided by the signal of the same reaction mix without compound.
Acknowledgments
This work was in part supported by University of Messina Research&Mobility 2015 Project (project code
RES_AND_MOB_2015_DE_LUCA) and by a grant from the KU Leuven (Geconcerteerde Onderzoeksacties No. GOA/15/019/TBA).
The authors wish to thank Kristien Erven, Cindy Heens, and Kris Uyttersprot (Rega) for dedicated technical assistance
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